Future potentials for using osteogenic stem cells and biomaterials in orthopedics

Ideal skeletal reconstruction depends on regeneration of normal tissues that result from initiation of progenitor cell activity. However, knowledge of the origins and phenotypic characteristics of these progenitors and the controlling factors that govern bone formation and remodeling to give a functional skeleton adequate for physiological needs is limited. Practical methods are currently being investigated to amplify in in vitro culture the appropriate autologous cells to aid skeletal healing and reconstruction. Recent advances in the fields of biomaterials, biomimetics, and tissue engineering have focused attention on the potentials for clinical application. Current cell therapy procedures include the use of tissue-cultured skin cells for treatment of burns and ulcers, and in orthopedics, the use of cultured cartilage cells for articular defects. As mimicry of natural tissues is the goal, a fuller understanding of the development, structures, and functions of normal tissues is necessary. Practically all tissues are capable of being repaired by tissue engineering principles. Basic requirements include a scaffold conducive to cell attachment and maintenance of cell function, together with a rich source of progenitor cells. In the latter respect, bone is a special case and there is a vast potential for regeneration from cells with stem cell characteristics. The development of osteoblasts, chondroblasts, adipoblasts, myoblasts, and fibroblasts results from colonies derived from such single cells. They may thus, theoretically, be useful for regeneration of all tissues that this variety of cells comprise: bone, cartilage, fat, muscle, tendons, and ligaments. Also relevant to tissue reconstruction is the field of genetic engineering, which as a principal step in gene therapy would be the introduction of a functional specific human DNA into cells of a patient with a genetic disease that affects mainly a particular tissue or organ. Such a situation is pertinent to osteogenesis imperfecta, for example, where in more severely affected individuals any improvements in long bone quality would be beneficial to the patient. In conclusion, the potentials for using osteogenic stem cells and biomaterials in orthopedics for skeletal healing is immense, and work in this area is likely to expand significantly in the future

In vitro and in vivo methods to determine the interactions of osteogenic cells with biomaterials

To assess new biomaterials for possible use as bone graft substitutes, a number of techniques allow interactions with osteoblastic cells to be studied, with respect to effects on proliferation and differentiation of osteoprogenitors. In vitro models include the use of bone explant cultures, fetal rat calvarial-derived osteoblast cells, primary stromal populations, transformed and non-transformed cell lines and immortalized osteoblast cell lines. However, these assessments are limited by the extent of osteogenic differentiation and bone formation that can be observed in vitro, species differences and phenotypic drift of cells cultured in vitro. The use of in vivo experimental systems such as the segmental/calvarial bone defect model, the subcutaneous implant model and the diffusion chamber implantation model circumvent some of these issues and, in the appropriate model, provide data on efficacy, biocompatibility and osteointegration of a biomaterial. The combination of in vitro and in vivo approaches together with the development of new cell labeling techniques, in particular the ability to genetically mark and select specific human bone cell populations provides new avenues for their potential evaluation in combination with appropriate biomaterials for clinical use. These in vitro and in vivo techniques are reviewed and those recently developed for assessment of human osteogenic cells should be applicable to many other cell systems where knowledge of specific human tissue or cell interactions with biomaterials is required

Skeletal progenitor cells and ageing human populations

1. Stem and progenitor cells present within bone marrow give rise to colony forming units-fibroblastic (CFU-F) which can differentiate into fibroblastic, osteogenic, myogenic, adipogenic and reticular cells. The decrease in skeletal bone formation and rate of fracture repair observed with ageing and in osteoporosis has been suggested to be due to a decrease in numbers of these progenitors, but human studies are limited. 2. We have tested the potential to form CFU-F in a total of 99 patients undergoing corrective surgery (16 controls, 14-48 years of age) or hip arthroplasty for osteoarthritis (57 patients, 28-87 years of age) or osteoporosis (26 patients, 69-97 years of age). Total colony number, alkaline phosphatase-positive colony number and colony size were determined. 3. No decrease in colony forming efficiency under the culture conditions used was observed in all populations examined irrespective of age, disease or gender, as determined by the lack of correlation between colony formation and age. 3. Examination of colony sizes showed a significant reduction in colony size with age in osteoarthritis and in control populations indicating a change in cellular proliferative potential with age. 4. Examination of number and percentage of alkaline phosphatase-positive CFU-F showed a significant decrease in osteoporotic patients compared with controls and osteoarthritis patients, indicating altered differentiation potential. 5. These results suggest that the reduction in bone mass with ageing may be due to reduction of the proliferative capacity of progenitor cells or their responsiveness to biological factors leading to alteration in subsequent differentiation. The maintenance of CFU-F number and alkaline phosphatase activity in these osteoarthritis patients may, in part, explain the inverse relationship observed for the preservation of bone mass between generalized osteoarthritis and primary osteoporosi

Effects of TGF? and bFGF on the differentiation of human bone marrow stromal fibroblasts

Adipocytes and osteoblasts have common origins from fibroblastic stem cells. Consequently, modulation of the processes of adipogenesis and osteogenesis has implications for the possible treatment of metabolic bone diseases, such as osteoporosis, in which medullary fat accumulates and trabecular bone volume decreases. It is likely that the balance between these two systems is affected by particular endogenous growth factors which are known to affect bone metabolism. We have therefore investigated the effects of transforming growth factor beta (TGF?), basic fibroblast growth factor (bFGF) and dexamethasone (Dex) on cultured human bone marrow (HBM) fibroblastic cells to observe the effects on adipogenesis and osteogenesis. In the absence of fetal calf serum (FCS), TGF? caused a dose-dependent increase in cell growth and alkaline phosphatase activity (AP); however, in the presence of FCS growth was inhibited at high concentrations and AP unaffected. TGF? increased matrix proteoglycan and collagen synthesis. bFGF inhibited AP and increased colony number and size, while Dex treatment increased AP activity and colony number, and both factors in combination resulted in an additive increase in growth. Dex-induced adipocyte formation was accelerated but not increased by bFGF. A significant inhibition of adipogenesis by TGF? was observed within 7 days. These results demonstrate the importance of biological factors known to be involved in bone remodelling in the regulation of osteogenesis and adipogenesis

Effects of beta mercaptoethanol on the proliferation and differentiation of human osteoprogenitor cells.

Antioxidants are known to influence metabolism and promote cell survival in a number of cell culture systems. However, their effects on the modulation of bone cell differentiation in vitro are not clearly defined. In the present studies we have investigated the effects of beta-mercaptoethanol (beta ME) and ascorbate alone and in combination on human osteoprogenitors derived from bone marrow fibroblasts. In primary marrow cultures, beta ME stimulated colony formation (2-fold), alkaline phosphatase activity (3.5-fold) and, increased DNA synthesis (8-fold) after 21 days. Cell proliferation was increased significantly by beta ME during the first 4 days of a 10-day culture period, indicating stimulation of marrow osteoprogenitor proliferation. Ascorbate did not significantly augment the effects of beta ME in primary cultures or long-term cultures of passaged bone marrow fibroblasts. These findings indicate a potential beneficial role for beta ME addition for the optimal maintenance of colony formation, cell proliferation and differentiation of marrow osteoprogenitor cells in primary human bone marrow fibroblast cultures

Modulation of osteogenic differentiation in human skeletal cells in Vitro by 5-azacytidine

Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system

Interconversion potential of cloned human marrow adipocytes in vitro

Information on the interconversion potential of adipocytes and other end cells characteristic of the stromal fibroblastic cell lineages, key in the understanding of bone turnover in metabolic diseases such as osteoporosis, is limited. The object of the present study was: i) to isolate relatively pure populations of adipocytes from human bone marrow; ii) to clone single adipocytes from these populations; and iii) to examine in vitro the interconversion potential of the progeny of these single-cloned adipocytes between the osteogenic and adipogenic phenotypes. Adipogenic colonies were isolated from the low-density floating fraction of normal bone marrow cells cultured in adipogenic media for 4 days. Single adipocytes were isolated and cloned by limiting dilution. Cloned adipocytes were found to dedifferentiate into fibroblast-like cells, and subsequently to differentiate into two morphologically distinct cell types: osteoblasts and adipocytes in appropriate culture systems. The adipocytic phenotype was confirmed by morphology, oil red O staining, and immunocytochemistry using antiserum to aP2. The osteogenic phenotype was confirmed by alkaline phosphatase, osteocalcin immunostaining using specific osteocalcin antiserum, and formation of mineralized cell aggregates. These findings demonstrate the extent of plasticity between the differentiation of adipocytic and osteogenic cells in human bone marrow stromal cell cultures. We have shown the ability of isolated clonal adipogenic cells to redifferentiate into cells of the osteogenic and adipogenic lineage and the interconversion potential of human marrow stromal cells in vitro. These results provide further evidence that the osteogenic and adipogenic cells share a common multipotential precursor

Modulation of osteogenesis and adipogenesis by human serum in human bone marrow cultures

Knowledge of the controlling mechanisms of human osteoprogenitor cell differentiation has important implications for understanding bone turnover. The in vitro differentiation of human bone marrow fibroblasts into adipogenic and osteogenic cells and the interaction of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone in this process has been investigated together with the effects of human serum. Marrow fibroblasts cultured in human serum and dexamethasone for 28 days, generated lipid containing cells as confirmed by morphology, Oil red O staining and immunocytochemistry using antiserum to the adipocyte-specific protein, adipocyte P2 (aP2). In cultures containing 1,25(OH)2D3 and dexamethasone, adipogenesis was stimulated within 21 days. Osteocalcin expression, as assessed by in situ hybridization, was dependent on the presence of 1,25(OH)2D3 and was decreased in cultures treated with dexamethasone. Northern analysis confirmed the decrease in osteocalcin expression and increase in lipoprotein lipase expression with the appearance of the adipogenic phenotype in these cultures. Marrow cultures maintained for 14 days in human serum and osteotropic agents before switching to fetal calf serum indicated the continuous requirement of human serum in these cultures for adipogenesis. These results demonstrate that human serum contains factors that exert dramatic effects on human bone marrow cell differentiation to augment the osteogenic and adipogenic activity of 1,25(OH)2D3 and dexamethasone

Effects of novel calcium phosphate cements on human bone marrow fibroblastic cells

The identification and characterization of biocompatible materials that augment bone cell proliferation and osteogenic activity have important therapeutic implications in skeletal reconstruction and joint replacement. In the present study, we have examined the effects of three biocements, biocement H, calcium-deficient apatite; biocement F, apatite + CaHPO(4); biocement D, carbonated apatite + CaHPO(4) + CaCO(3) and an amorphous calcium phosphate (ACP) proposed as implant fixing materials, on the growth, differentiation, and cell surface interaction of human bone marrow fibroblastic cells. These cells are known to be progenitors of osteoblasts, chondroblasts, adipocytes, myoblasts, and reticulocytes. Alkaline phosphatase enzyme activity, a marker of the osteoblast phenotype, was increased by a factor of two- to sixfold on carbonated apatite, one- to sixfold on apatite and three- to 10-fold on calcium-deficient apatite, over levels observed on plastic. Cell proliferation was significantly reduced. Photomicroscopic examination indicated high biocompatibility with close adhesion of the bone marrow fibroblastic cells to composites D, F, and H. Longer term marrow cultures (15 days) confirmed the stimulation of cell differentiation, as assessed by collagen production, over cell proliferation, of cells grown on carbonated apatite. Enhanced osteoblastic differentiation was observed on a 70% carbonated apatite, which has a composition similar to bone mineral, whereas cell toxicity was observed on cells grown on amorphous calcium phosphate. This in vitro human bone marrow fibroblast culture system provides a simple and effective method for the evaluation of new biomaterials. The development of these novel cements may be of potential use in orthopedic implant